Iron-deficiency anemia increases intestinal bacterial translocation in rats

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Critical Care Volume 9 Suppl 2, 2005

Third International Symposium on Intensive Care and Emergency Medicine for Latin America São Paulo, Brazil, 22–25 June 2005 Published online: 9 June 2005 These abstracts are online at http://ccforum.com/supplements/9/S2 © 2005 BioMed Central Ltd

Basic science

Figure 1

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Introduction Neutrophils have been involved in sepsis-induced organ damage. Neutrophils could be directly activated by TLR binding ligands including LPS. IRAK-1 is one of many intracellular proteins that are activated upon stimulation of TL receptors. This triggers a series of events that results in the migration of NF-κB into the nucleus and the activation NF-κB-dependent genes. Objectives To identify a single nucleotide polymorphism at position 532 (coding SNP) in volunteers and patients with sepsis. To determine whether IRAK-1SNP532 results in a decrease in neutrophil NF-κB activation in volunteers and patients with sepsis. To evaluate neutrophil gene expression patterns in IRAK-1SNP532 and wildtype patients with sepsis. Methods Thirty severe sepsis patients and 34 healthy volunteers were enrolled in this study. Peripheral blood was obtained and neutrophils were isolated by plasma–percoll gradients after dextran sedimentation of erythrocytes. Neutrophils from volunteers were resuspended in RPMI and cultured with or without 100 ng/ml LPS for 60 min. The electrophoretic mobility shift assay technique was used to measure the NF-κB activation. Real-time PCR allelic discrimination assays were developed by the assay-by-design service offered by Applied Biosystems (Foster City, CA, USA). Probe and primer combinations were designed at the single nucleotide polymorphism 532. PCR reactions were performed according to the manufacturer’s manual using the Applied Biosystems 7500 Real-Time PCR system. Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after LPS stimulus. Results The median AUC for NF-κB activation was higher in wildtype genotyped neutrophils as compared with IRAK-1SNP532 genotyped neutrophils (85.2 vs 100.5, P = 0.05) (Fig. 1). In terms of kinetics pattern, we found some differences on nuclear levels of NF-κB in neutrophils from volunteers cultured with LPS. At 30 min after LPS, the culture nuclear translocation of NK-κB was significantly greater in wildtype genotyped neutrophils than in IRAK-1SNP532 genotyped neutrophils. Even after 60 min, the NF-κB translocation remained high in wildtype genotyped neutrophils, while in IRAK-1SNP532 genotyped neutrophils the NF-κB translocation was similar to baseline (Fig. 2). In unstimulated neutrophils from septic patients, the NF-κB translocation was significantly lower in IRAK-1SNP532 genotyped neutrophils than in

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J Arcaroli, E Silva, Q He, D Svetkauskaite, C Coldren, J Maloney, JS Park, E Abraham Division of Pulmonary and Critical Care Medicine, University of Colorado, Denver, Colorado, USA Crit Care 2005, 9(Suppl 2):P1 (DOI 10.1186/cc3545)

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κB and expression of related genes Decreased activation of NF-κ in IRAK-1SNP 532 neutrophils from volunteers exposed to endotoxin and in unstimulated neutrophils from septic patients

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wildtype genotyped neutrophils (1.20 vs 2.10, P = 0.05) (Fig. 3). Finally, the expression of some inflammatory related genes (IL-8, IL1β, MIP-2, COX-2, and SOD2) was decreased in IRAK-1SNP532 genotyped neutrophils. Conclusion IRAK-1SNP532 genotyped neutrophils from volunteers (after LPS ex vivo challenge) and from septic patients are associated with lower NF-κB activation and lower expression of some IRAK1-related genes. These results demonstrate that IRAK1

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Third International Symposium on Intensive Care and Emergency Medicine for Latin America

plays a critical role in the inflammatory response and, potentially, a polymorphism in IRAK1 may alter the immune response impacting clinical outcome.

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Septic patients HMGB1

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κB activation after Gene expression and intracellular NF-κ HMGB1 and LPS stimuli in neutrophils from septic patients E Silva, J Arcaroli, Q He, D Svetkauskaite, C Coldren, J Nick, K Poch, JS Park, E Abraham Division of Pulmonary and Critical Care Medicine, University of Colorado, Denver, Colorado, USA Crit Care 2005, 9(Suppl 2):P2 (DOI 10.1186/cc3546)

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Introduction Neutrophils play a major role in sepsis-induced organ dysfunction, especially in the lung. HMGB1 has emerged as a late cytokine and is implicated in the perpetuation of inflammatory stimulus and organ dysfunction development as well. There are limited data about neutrophil response patterns to HMGB1 in septic patients, and whether those patterns could be different from those following LPS exposure. Objectives To evaluate the differences of gene expression and activation of NF-κB, Akt, and p38MAPK in blood neutrophils from septic patients exposed to HMGB1 and LPS; and to compare response patterns between blood neutrophils from patients and healthy volunteers. Methods Twenty-two sepsis-induced acute lung injury patients and 34 healthy volunteers were enrolled in this study. The primary clinical variables collected were the 28-day survival and the presence of shock at ICU admission. Peripheral blood was obtained and neutrophils were isolated by plasma–percoll gradients after dextran sedimentation of erythrocytes. Neutrophils were resuspended in RPMI and cultured with or without 1000 ng/ml rHMGB1 or with or without 100 ng/ml LPS for 15, 30, and 60 min. The electrophoretic mobility shift assay technique was used to measure the NF-κB translocation, while western blot analysis was used to determine Akt phosphorylation and an ELISA was used to determine p38MAPK phosphorylation. Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after either HMGB1 stimulus or LPS stimulus. P < 0.05 was considered significant. Results Although with some similarities, HMGB1 and LPS induced distinct patterns of gene expression in peripheral blood neutrophils from septic patients. A Venn diagram (Fig. 1) displays genes upregulated greater than twofold that are both common and unique after both stimuli. Using functional ontology, the genes upregulated by both HMGB1 and LPS primarily consisted of cytokines, chemokines, coagulation-related proteins, phosphatases, and transcriptional regulators factors. Importantly, while HMGB1 induced an HMGB1-related gene downregulation, LPS did not induce any changes in HMGB1 gene expression in these patients. Regarding intracellular activation, both HMGB1 and LPS increased translocation of NF-κB and the phosphorylation of Akt and p38MAPK in neutrophils from septic patients. However, there were some differences in terms of the degree and kinetics of activation between neutrophils cultured with LPS and HMGB1 (Fig. 2). There are no important differences in terms of intracellular activation when we compared neutrophils from septic patients with those from volunteers. Finally, neither NF-κB translocation nor kinase phosphorylation was associated with sepsis severity. However, the majority of genes in unstimulated neutrophils and after HMGB1 had a higher expression in mild patients. In contrast, CCL20, CCRL2, CIAS1, PTGER, PTX3, and MAP3K8 had a higher expression in severe patients only after LPS stimulus.

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Conclusion Although with some similarities, HMGB1 and LPS induced distinct pattern of gene expression in neutrophils from septic patients. Both stimuli were able to increase intracellular activation and this activation was similar to that found in neutrophils from volunteers, showing that even after sepsis stimulus the neutrophil keeps its ability to respond to a second hit. P3 Macrophage chemoattractant protein 1 and outcome in cardiopulmonary bypass KC Pereira, HF Mendonça-Filho, GS Gomes, M Fontes, MLAF Mendonça, PMM Nogueira, HFR Dohmann Translational Research, Pró-Cardíaco Hospital, Rio de Janeiro, Brazil Crit Care 2005, 9(Suppl 2):P3 (DOI 10.1186/cc3547) Introduction Cardiopulmonary bypass (CPB) is associated with systemic inflammation that involves a number of cytokines, and, despite scarce data, macrophage chemoattractant protein 1 (MCP-1) could be implicated in postoperative organ dysfunction. This pilot study attempted to describe perioperative circulating

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levels of MCP-1 and to investigate possible correlations with the intensity of postoperative organ dysfunction. Methods Under informed consent, 20 patients submitted to cardiac surgery with CPB were consecutively studied. MCP-1, macrophage migration inhibitory factor (MIF), IL-6 and IL-10 were assayed by ELISA in peripheral blood sampled at anesthesia induction and 3, 6, 10 and 24 hours post-CPB. Data were analyzed by ANOVA for repeated measures with the Bonferroni test, and the two-tailed Spearman test for correlations with postoperative outcomes, as measured by the multiple organ dysfunction score at the third postoperative day (MODSd3). Significance was assumed for P < 0.05. Results Similar to MIF and IL-6, blood levels of MCP-1 significantly changed after CPB. From baseline levels (69.44 ± 15.92 pg/ml), MCP-1 reached peak values 3 hours post-CPB (387.11 ± 108.87 pg/ml), and progressively declined thereafter. MODSd3 was associated with the levels of MCP-1 measured at anesthesia induction (P = 0.010, rho = 0.606) and at 6 hours post-CPB (P = 0.037, rho = 0.508). Levels of IL-6, 6 hours post-CPB, were also associated with MODSd3 (P = 0.008, rho = 0.616). Conclusion CPB-induced levels of MCP-1 and IL-6 were related to postoperative outcome. Additionally, preoperative levels of MCP-1 were also related to postoperative outcome. Although of limited sample size, these findings can stimulate further studies to explore the role of MCP-1 in the prediction of, and also as a potential therapeutic target in, post-CPB organ failure.

P4 Macrophage migration inhibitory factor as a diagnostic tool for acute coronary syndrome KC Pereira, M Viegas, G Gomes, NV Gomes, A Potch, BR Tura, HTF Mendonca-Filho Pró-Cardíaco Hospital, Rio de Janeiro, Brazil Crit Care 2005, 9(Suppl 2):P4 (DOI 10.1186/cc3548) Introduction Inflammatory activity is recognizably enrolled in the physiopathological basis of acute coronary syndrome (ACS). Considering the diagnostic challenge related to ACS when typical electrocardiographic (EKG) findings are absent, we evaluated the role of migration inhibitory factor (MIF), soluble CD40 ligand (CD40L) and IL-6 in this scenario. Design A prospective, observational, cohort pilot study. Setting The emergency division at a tertiary care cardiology center. Methods Under informed consent, patients whose main complaint was chest pain were considered eligible. Exclusion criteria consisted of associated neoplastic, infectious or inflammatory disease as well as EKG with ST-segment elevation above 1 mm. Within the first 12 hours of admission, venous blood was sampled for sCD40L, MIF and IL-6 assays (ELISA-sandwich; R&D Systems, Minneapolis, MN, USA). A high risk for ACS was defined by nuclear image, angiographic and/or enzymatic criteria (troponin I >1.0). Results From 195 patients included, 69 (35.4%) were considered under high risk for ACS and 126 (64.6%) as non-ACS patients. Within the overall patients, a positive bidirectional correlation was observed between sCD40L and MIF, and a negative correlation was observed between each of these and IL-6. After principal component analysis, non-parametric tests showed significant differences between the two groups concerning levels of MIF (P < 0.0001) and IL-6 (P = 0.012). For discrimination of patients under high risk for ACS, areas under the receiver operator curves for MIF and IL-6 were 0.69 and 0.61, respectively.

Conclusion In spite of complex interactions among inflammatory mediators, levels of MIF are independently related and possibly have a role in the identification of patients under high risk for ACS among those with chest pain without ST-segment elevation. Further studies are needed to explore MIF potential as a new diagnostic tool in ACS.

P5 α in pediatric Study of polymorphisms in the genes for TNF-α patients in the ICU of Instituto Fernandes Figueira — Fundação Oswaldo Cruz MA Azevedo, GI Matos, SCM Sales, EP Sampaio, PG Elsas, M Moraes, MIG Elsas ICU of Instituto Fernandes Figueira – Fundação Oswaldo Cruz, Rio de Janeiro, Brazil Crit Care 2005, 9(Suppl 2):P5 (DOI 10.1186/cc3549) Recent advances in molecular biology and genomics have led to increased understanding of the pathophysiological process directly relevant for pediatric intensive care, such as sepsis, ARDS and multiple system organ failure. Several point mutations, called single nucleotide polymorphisms (SNPs), have been identified. The gene of the inflammatory cytokine TNF-α has been indicated as an important candidate for such studies since it has an important function on immuno-inflammatory response, and important SNPs have already been identified. Among these, those that can be detached are those located at positions –863 (C→A) and –308 (G→A) in its promoter region, which can be related directly with the expression of this cytokine and, consequently, with the regulation of the circulating levels of the protein. The aim of this study was to evaluate the polymorphism of the TNF-α gene in positions –863 and –308 in the group of pediatric patients with sepsis/SIRS in the ICU of the Fernandes Figueira Institute — FIOCRUZ. The DNA was extracted from samples of whole blood and with swabs containing oral mucosal cells (in patients that had received blood transfusion) with a mixture of detergents. The molecular determination of the genotypes was carried out using the techniques of PCR-restriction fragment length polymorphism for position –308 and PCR-amplification refractory mutational system for position –863. Eighty-five patients of both sexes and with age varying between 0 and 12 years, with sepsis/SIRS, were admitted. The genotypic frequencies were GG (0.8) and GA (0.2) for –308, and CC (0.77), CA (0.22) and AA (0.01) for –863. The allelic frequency for –308 was G (0.9) and A (0.1), while for –863 it was C (0.88) and A (0.12). Through the analysis of the possible genotypic combinations, it was observed that the more frequent haplotype was CG (0.78) and, using analyses of maximum likelihood, it was verified that the locus did not meet in linkage disequilibrium. The population in this study was in Hardy–Weinberg equilibrium for both the studied polymorphisms. The present study evaluated the genetic characterization of locus TNF-α in this population of pediatric patients with sepsis/SIRS of the metropolitan region of Rio de Janeiro, being comparable with population data of studies in other countries. However, other studies using controls groups should be performed to verify the utility of these polymorphisms as molecular markers for sepsis severity or susceptibility.

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Cardiology P6 Electrocardiographic disturbances after cardiac surgery in patients with coronary disease HP Guimarães, RD Lopes, MPF Costa, PHR Leal, AP Resque, F Freitas, LO Bueno, FR Machado, JLG Amaral Federal University of São Paulo, UNIFESP, EPM, São Paulo, Brazil; São Camilo University Center, São Paulo, Brazil Crit Care 2005, 9(Suppl 2):P6 (DOI 10.1186/cc3550) Introduction The risk of mayor cardiovascular events in the postoperative period can reach up to 25%, particularly if there were CAD antecedents. Methods A prospective study in patients with CAD diagnosed by complementary examination (arteriography) or clinical history (MI, angina), monitored in the first 24 hours of the postoperative period, using the system of continuous electrocardiography monitoring (Holter®) of three canals and myocardial injury markers (CPK, MB and troponin). Results Initial inclusions involved seven (70%) male patients and three (30%) female patients; the age varied from 48 to 90 years, with an average of 66 years and a mode of 56 years. The APACHE varied score from 4 to 14 with a mode of 14, and the median risk of death was 5%. Three patients (30%) had antecedents of coronary artery bypass graft and three (30%) patients of angioplasty; four patients had recent coronary arteriography with injuries of up to 60% in up to two vases. Arrhythmias had been present in all the patients, being supraventricular extrasystole and supraventricular tachycardia in five (50%) patients and ventricular extrasystole in five (50%) patients. Three (30%) patients had presented silent myocardial ischemia with disturbances in the ST segment varying from 2.0 to 3.4 mm in at least two canals, with positive trooping in two (20%) patients, although an echocardiogram of stress with dobutamine (previous to surgery) was negatively affected in two of these patients. We did not have death occurrence, having only prolongation of hospital internment in the three patients with myocardial ischemia. Conclusion The occurrence of myocardial ischemia in patients with CAD can be a frequent event and, despite evaluation daily post surgery, must always rigorously be monitored. Reference 1.

Guimarães HP, et al.: Alterações Eletrocardiográficas Durante a Retirada da Ventilação Mecânica: Resultados iniciais da Monitoração Eletrocardiográfica Contínua (Holter®). Rev Brasil Terapia Intensiva 2002, 99(suppl 1):P423.

P7 Clinical impact of atrial electric stabilization in patients with chronic atrial fibrillation undergoing cardiac surgery W Homena Jr, M Padilha, D Moreira, B Santos, F Gouvea, JA Pedrosa, G Silva, L Alves, JO Brito, R Gomes Instituto Nacional de Cardiologia de Laranjeiras, Rio de Janeiro, Brazil Crit Care 2005, 9(Suppl 2):P7 (DOI 10.1186/cc3551)

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Introduction Atrial fibrillation (AF) is frequent in patients undergoing cardiac surgery (CS). Despite the high prevalence of chronic AF in patients with valvular heart disease, few studies have assessed the clinical evolution of these patients when undergoing CS. Objective To assess the clinical outcome of patients with chronic AF undergoing CS who evolved with electric atrial stabilization in the immediate postoperative period (IPO).

Methods A prospective and observational study of patients undergoing CS with extracorporeal circulation (ECC) divided into two groups: Group 1 (G1), patients who maintained AF; and Group 2 (G2), patients who reverted to AF in the IPO. The following preoperative (PRE) parameters were assessed: left ventricular (LV) and right ventricular function; functional class; left atrium (LA) size (>5 mm); LV hypertrophy (>12 mm); presence of SAH, DM, COPD, CAD; use of AA drugs; and LBBB. The following perioperative (PER) parameters were assessed: atrial thrombus; plication of the atrial auricle; time of ECC and of anoxia; and chemical and/or electric CV. The following variables influenced the clinical outcome: mechanical ventilation time (MVT), ICU length of stay (ICULOS), hospital length of stay (HLOS), and maintenance of AF. The statistical analysis involved the following tests: Student t test, Fisher exact test, and Mann–Whitney test. Results G1 comprised 21 patients (14 women, 66.6%) with a mean age of 52.6 years, and G2 comprised 33 patients (15 women, 45%) with a mean age of 49.8 years (P = not significant). No statistical difference was observed in regard to the PRE and PER variables, except for the LA size >5 mm (G1 85.7%, G2 45%, P = 0.0031), MVT, ICULOS, and HLOS. Of G1 patients, only one (4.7%) reverted his rhythm to sinus rhythm, while 24 patients (72.7%) in G2 maintained their sinus rhythm until ICU discharge (P = 0.000022). Conclusion In this sample, LA size was the major predictor of maintenance of AF, which did not determine greater morbidity. Once AF is reverted, however, one should not restrain efforts to maintain atrial electric stability. P8 Clinical impact of the prophylactic use of intra-aortic counterpulsation in high-risk patients undergoing myocardial revascularization W Homena Jr, JA Albuquerque, D Moreira, B Santos, A Weksler, R Vegni, A Pontes, S Olival, L Alves, FR Gouvea, JO Brito, RV Gomes Instituto Nacional de Cardiologia de Laranjeiras, Rio de Janeiro, Brazil Crit Care 2005, 9(Suppl 2):P8 (DOI 10.1186/cc3552) Introduction The use of the intra-aortic balloon (IAB) has been well established in the clinical management of patients with problems such as refractory myocardial ischemia, cardiogenic shock, and difficulty in weaning from extracorporeal circulation (ECC). However, the literature lacks evidence supporting the ‘prophylactic’ use of the IAB in high-risk patients undergoing myocardial revascularization (MR). Objective To assess the clinical outcome of surgical high-risk (HR) patients undergoing MR, who received a ‘prophylactic’ IAB. Methods A prospective and observational study of a population undergoing elective MR. High-risk patients were defined as those having severe LV dysfunction (EF 50% of the luminal diameter). The sample was divided into two groups: Group 1 (G1) with ‘prophylactic’ IAB, and Group 2 (G2) without IAB. The influence of the following variables on clinical outcome was assessed: use of amines; fluid balance (FB) in the perioperative period (PER); time of ECC, anoxia, and mechanical ventilation (MVT); ICU length of stay (ICULOS); hospital length of stay (HLOS); complications of the procedure; and death. Results G1 comprised 16 patients (87.5% men) with a mean age of 61.6 (SD 8.6) years, and G2 comprised 39 patients (87.1% men) with a mean age of 56 (SD 8.0) years (P = not significant). No difference was observed between the groups regarding the

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other base variables, except for BMI (P = 0.00035). In regard to clinical outcome, only FB in the PER (G1 median 1695 ml, interquartile interval [IIQ] 923–1865; G2 median 2061 ml, IIQ 1257–2860, P = 0.03) and MVT (G1 median 11.5 hours, IIQ 7–26 hours; G2 median 8 hours, IIQ 5–12 hours) had statistical significance. No significance was observed regarding the use of amines, time of ECC, ICULOS, HLOS, and death. No complications inherent to IAB use were observed. Conclusion The ‘prophylactic’ use of the IAB showed no benefit regarding morbidity and mortality in the population studied. The greater blood volume replacement and prolonged MVT emphasize the need for care when indicating this procedure. P9 Neurologic complications in cardiac surgery: can risk scores be applied? W Homena Jr, D Moreira, B Santos, M Nolasco, A Weksler, S Olival, R Vegni, A Pontes, L Alves, JO Brito, RV Gomes Instituto Nacional de Cardiologia de Laranjeiras, Rio de Janeiro, Brazil Crit Care 2005, 9(Suppl 2):P9 (DOI 10.1186/cc3553) Introduction Neurologic complications (NC) in cardiac surgery are not rare (5–15%). Their etiopathogeny is multifactorial, and the risk factors are numerous. Neurologic complications result in high morbidity and mortality rates, and high hospital costs. Most risk scores assess mortality, and the risk for stroke assessed by the AHA/ACC score refers only to patients with coronary disease. One may thus question whether risk scores for NC can be applied in a general population. Objective To assess the risk scores of patients with NC undergoing cardiac surgery. Methods A retrospective observational study including information about 1431 patients from a databank, of whom 45 (3.1%) had reversible or permanent neurologic deficit. The sample was divided into two groups: Group 1 (G1), patients with NC; and Group 2 (G2), the historic control. The Cleveland score, Euroscore, and AHA/ACC score for stroke were assessed, as was the occurrence of death. The Student t test was used for analyzing the means of continuous variables. Results G1 comprised 24 men (53.3%), and the mean age of patients was 63.5 (SD 13.6) years. The surgeries were as follows: 26 myocardial revascularizations (57.7%), 12 valvular replacements (26.6%), one combined (2.2%), two congenital (4.4%), and three aortic surgeries (6.6%). The means of the Cleveland score, Euroscore, and AHA/ACC score were: in G1: 4.5 (SD 3.3), 6.1 (SD 4.2), and 4.1 (SD 2.6), respectively; and in G2: 2.9 (SD 2.6), 3.6 (SD 2.8), and 2.5 (SD 2.5), respectively, with statistical significance (P < 0.0001, P < 0.0001, and P < 0.0001). The mortality rate was 24.4% in G1 and 9.2% in G2 (P = 0.002). Conclusion The risk scores for cardiac surgery applied for mortality reflected a greater incidence of neurologic complications in this population. P10 Cardiogenic shock: an experimental animal model C Tanamati, M Monachini, M Cantarelli, PV Khouri, GA Amarante, P Martins, F Coelho, G Schettino Hospital Sírio Libanês, São Paulo, Brazil Crit Care 2005, 9(Suppl 2):P10 (DOI 10.1186/cc3554) Objective To create an experimental animal model of cardiogenic shock for learning and to test new therapeutic strategies.

Methods Adult white pigs (70 kg) received both intravenous anesthesia (acepromazine 0.3 mg/kg, midazolam 0.2 mg/kg, fentanyl 250 µg/kg, thiopental sodium 12.5 mg/kg and pancuronium 0.4 mg/kg) and inhaled anesthesia (halothane 1%), and were intubated and mechanically ventilated. An arterial line was obtained through dissection and puncture of the common femoral artery. A continuous cardiac output catheter (Edwards Lifescience, USA) was introduced through the dissected internal jugular vein and was positioned using the arterial pulmonary pressure curve, allowing monitoring of the right atrial pressure, pulmonary artery pressure, pulmonary wedge pressure (PAop) and SvO2. Through median sternotomy, the pericardium was opened longitudinally and the heart was exposed. The baseline ECG and hemodynamic data were recorded and after a 6-0 polypropylene suture was passed under the proximal anterior descending coronary artery that was snared for up to 10–15 min. An ECG was then obtained to show typical ischemic alterations, and a regional myocardium color change and regional myocardial hypocontractility were observed. The presence of cardiogenic shock was defined by cardiac output index 20 mmHg and mean arterial pressure